UNIVERSITY OF GONDAR

COLLEGE OF MEDICINE AND HEALTH SCIENCES

SCHOOL OF PHARMACY
DEPARTMENT OF PHARMACOGNOCY

Evaluation of In Vitro Antibacterial Activities of the Root Extracts of

Cucumis ficifolius A. Rich (Cucurbitaceae)

A Directed Study Paper Submitted to the School of Pharmacy, College of

Medicine and Health Science, University of Gondar in Partial Fulfilment
for the Requirements of Bachelor Degree in Pharmacy

By:
Samuel Agegnew and Fraesenay Mezmur

Advisors:
Mr. Yohannes Kelifa (B. Pharm, MSc)
Mr. Abyot Endale (B. Pharm, MSc, Asst. Prof)

JUNE, 2018
GONDAR, ETHIOPIA
Abstract
Background: Infectious diseases are becoming major health concern. One of the main causes
of this problem is the widespread emergence of acquired bacterial resistance to antibiotics.
Antimicrobial resistance is harmful to mankind, because most of the infectious
microorganisms understand the mechanism of drug action and develop tolerance to it. The
current cost of most of the chemotherapeutic agents is unaffordable to the public especially in
developing countries. Discovery of new generation drugs against these infections from
natural products is highly desired for development of effective, affordable and safe
antibacterial agents that would be used as complimentary or alternative with convectional
medicines.

Objective: The overall objective of the study was to evaluate the antibacterial activity of
crude hydroalcolic root extract of Cucumis ficifolius A. Rich.

Methods: The roots of Cucumis ficifolius A. rich was collected and dried under the shade.
The dried plant material was pulverized and macerated in 80% methanol. The crude extract
was evaluated for the antibacterial activity against six standard bacteria species by agar well
diffusion methods. Preliminary photochemical analysis was conducted using standard
procedures.

Result: Reddish powder crude extract with 25% yield was obtained. Preliminary
phytochemical screening of the methanolic root extract of C. ficifolius revealed that the
presence of bioactive compounds such as alkaloids, terpinoid, flavonoids, phenols,
antraqunolones and steroids. In contrast, with its ethnobotanical claim for antimicrobial
activity, the crude extract of C. ficifolius did not show any significant inhibitory activity
against the tested bacteria strain in this study.

Conclusion: From the results of this study it can be concluded that phytochemical screening
of the root extract of Cucumis ficifolius A. rich indicated the possible presence of alkaloids,
steroid, terpinoid, flavonoids, phenols and antraqunolones. The presence of these
phytochemicals in this plant enhances their pharmaceutical and therapeutic potentials.
However, the antimicrobial results reported in this study doesn’t have a correlation between
the traditional uses which might be due to some factors like geographical and seasonal
variation during collection of this plant.

Keywords: Antibacterial activity, Infectious disease, Cucumis ficifolius, A. Rich, Ethiopia.

I
Acknowledgements
First of all, we would like to thank almightily “GOD” the most gracious and merciful for his
kindness, love and help for our success and achievement. Our heart full great gratitude goes
to our advisors Mr.Yohannes Kelifa and Mr. Abyot Endale for their invaluable advice,
support and unrelenting encouragement. We wish God to give them the fruits of life. We also
want to extend our acknowledgment to Mr. Gashaw Sisay and Mr. Endeshaw Demssie the
laboratory assistants and all other people who support us technically and morally. Last but
not least our finial gratitude goes to Mr. Getenet, lecturer of Microbiology, and Mr. Azanew,
microbiology laboratory technician for their invaluable support and guidance during
antibacterial activity test.

II
Table of contents

Abstract ...................................................................................................................................... I
Acknowledgements ................................................................................................................... II
List of tables .............................................................................................................................. V
List of figures .......................................................................................................................... VI
List of abbreviations and acronyms ....................................................................................... VII
1 Introduction ..........................................................................................................................1
1.1. Background .........................................................................................................................1
1.2. Statement of the problem ....................................................................................................2
1.3. The role of phytochemicals in management of bacterial infections ...............................2
1.4. The family Cucurbitaceae .................................................................................................3
1.5. The Genus Cucumis ............................................................................................................4
1.5.1. Pharmacological activity of genus Cucumis ....................................................................5
1.5.2.Isolated compound ............................................................................................................6
1.6. Cucumis ficifolius................................................................................................................8
1.6.1. Botanical Description.......................................................................................................8
1.6.2. Ethnobotanical uses .........................................................................................................8
1.6.3. Pharmacological activity..................................................................................................9
1.6.4. Significance of the study..................................................................................................9
2 Objectives ..........................................................................................................................10
2.1. General objectives ............................................................................................................10
2.2. Specific objectives ...........................................................................................................10
3 Material and Methods ........................................................................................................11
3.1. Chemicals ..........................................................................................................................11
3.2. Equipments and supplies...................................................................................................11
3.3. Plant materials ...................................................................................................................11
3.4. Bacterial strains .................................................................................................................11
3.5. Preparation of plant extract ...............................................................................................12
3.6. Determination of antibacterial activity .............................................................................12
3.7. Preliminary Phytochemical screening...............................................................................12
3.8. Ethical consideration .........................................................................................................14
4. Results ..................................................................................................................................15
4.1 Percentage yield of crude extract .......................................................................................15

III
4.2. Preliminary phytochemical screening .............................................................................15
4.3. Anti-bacterial activity .......................................................................................................16
5. Discussion ..........................................................................................................................17
6. Conclusion .........................................................................................................................18
7. Recommendation ...............................................................................................................19
8. References ..........................................................................................................................20
9. Annex .................................................................................................................................24

IV
List of tables
Table 1: Preliminary phytochemical screening results of the root extract of Cucumis ficifolius
.................................................................................................................................................. 15
Table 2: Antibacterial activity of crude root extracts of Cucumis ficifolius A. Rich ............. 16

V
List of figures
Figure 1: Phenylethyl isolated from the Cucumis melo ............................................................ 7
Figure 2: Sphingolipids Isolated from the Stems of Cucumis sativas L. .................................. 7
Figure 3: Morphological view of Cucumis ficifolius A. Rich................................................... 8

VI
List of abbreviations and acronyms
DMSO Dimethyl Suphoxide
DPPH Diphenyl picrylhydrazyl
INT Iodonitrotetrazolium Chloride
MHA Mueller Hinton Agar
RA Reference Antibiotic
UOG University of Gondar
WHO World Health Organization
ZI Zone of Inhibition

VII
1 Introduction

1.1. Background
Infection is the term that defines the entrance and development of infectious agent in a human
body or animal body, whether or not it develops into a disease (1). Infectious disease is
defined as a disease caused by a specific infectious agent or its toxic product that results from
transmission of that agent or its product from an infected person, animal, or reservoir to a
susceptible host, either directly or indirectly through an intermediate plant or animal host,
vector or inanimate environment (1). Some of pathogenic infectious agent include
Staphylococcus aureus (infections of skin, septic arthritis and food intoxication) (2), Bacillus
subtilis and Bacillus licheniformis (food intoxication) (3), Esherichia coli (foodborne illness,
diarrhea) (4). Salmonella enterica and Salmonella typhimirium (food intoxication) (5,6).

The development of pathogenesis of infectious diseases into the host body involves different
periods of time (7) between the host becoming infected and developing the disease or
becoming a new transmitter of the agent. These different periods includes; Incubation period
is the time interval between the effective exposure of the susceptible host to an infectious
agent and the appearance of signs and clinical symptoms; latent period is the time period
from infection to onset of the ability to infect; Prodromal period is the time between the
insight of illness by the host and the appearance of signs and symptoms based on a clinical
diagnosis of the disease; communicable period is the time interval during which the infected
host eliminates an agent to the environment and new susceptible individuals can become
infected(1)

The epidemiology of infections has significantly changed in the last 30 years. As a result,
bacterial illnesses are currently considered as emerging diseases. World Health Organization
(WHO) reports that infectious diseases are responsible for over 50% deaths worldwide,
occurring mainly in tropical and developing countries (8).

Infectious diseases constituted the most serious health issue in the world until the beginning
of the 20th century when chronic degenerative diseases began to dominate this scenario in
developed countries (1). Presently, they are on the rise because of antimicrobial resistance
(9), and are more frequent in developing countries where one out of two people is dying
prematurely from infections, when compared to developed countries (10).

1
 1.2. Statement of the problem
To date even though a wide range of synthetic and semi-synthetic antibacterial agents is
available for the control of infectious diseases (11); resistance of bacteria to the available
antibiotic agent is a growing and worrisome problem that continues to challenge both
developing and developed countries (12). Although health care providers treat infected
patients with narrow and broad spectrum antibacterial drugs, the use of these synthetic drugs
may subject the patient to a higher risk, due to the possibility of the occurrences of more
harmful adverse effects (12). Consequently, infectious disease complications remain an
important cause of mortality and morbidity among hospitalized patients(12).

Antimicrobial resistance is harmful to mankind, because most of the infectious

microorganisms understand the mechanism of drug action and develop resistance to it (13).
Due to these negative effects, and the constant development of bacterial resistance, there is a
continuous need to develop newer antimicrobial agents effective against microorganisms and
less harmful to the host (11). Moreover, the current cost of most of the chemotherapeutic
agents is unaffordable to the public especially in developing countries (14). Numerous
authors have therefore explored the activity of plant extracts, fractions and pure compounds
contained in the extracts relevant bacterial infections in Asia and in Africa (15).

Due to restricted access to proper medicine, in many developing countries, people are still
using plants to treat the most prevalent infections(16). Hence, plant derived antimicrobials
have received considerable attention in recent years. Therefore, discovery of new generation
drugs against these infections from natural products is highly desired for development of
effective, affordable and safe antibacterial agents that would be used as complimentary or
alternative with convectional medicines.

1.3. The role of phytochemicals in management of bacterial infections

The medicinal and pharmaceutical properties of plants are due to the type of chemical
substance they produce and store. These include compounds that are utilized as food by man
and other animals and also other compounds that exert physiological effects on them. This
second group of chemical substances often referred to as secondary metabolites, give plants
their therapeutic properties (17).

Phytochemicals (from the Greek word phyto, meaning plant) are biologically active, naturally
occurring chemical compounds found in plants, which provide health benefits for humans
further than those attributed to macronutrients and micronutrients (18). Phytochemicals

2
obtained from natural products have been used worldwide in traditional medicine since
antiquity and area source of potential and powerful drugs (11). For thousands of years,
natural products have been used in traditional medicine all over the world and predate the
introduction of antibiotics and other modern drugs (19). Since, medicinal plants constitute a
wide variety and diversity of secondary metabolites, medicinal plants could be used as a good
source of antibacterial agents (20). Natural products, either pure compounds or standardized
plant extracts, provide unlimited opportunities for novel and suitable additives and drug
treatments because of their constituent a wide range of chemical diversity (21). Usage of
plant-derived antimicrobial agents might be effective in reducing the dependence on
antibiotics and minimizing the chances of antibiotic resistance in foodborne pathogenic
microorganisms (22).

Phytochemicals found in plants such as tannins and alkaloids, which have been found in vitro
to have antimicrobial properties (23). In addition, polyphenolic biomolecules have been
expected to play important roles in creating new and better therapeutic agents (24). Indeed,
the flavonoids can be inhibited the nucleic acid synthesis, cytoplasmic membrane function,
and can act on energy metabolism (25). Flavonoids include flavonols, flavones, isoflavones,
flavanones, anthocyanidins most commonly known for their antioxidant and antimicrobial
biological activities (26,27).

1.4. The family Cucurbitaceae

The family Cucurbitaceae is also called vine family which consists of 825 species under 125
genera (28). The major phytocompounds present are the phytochemicals like glycosides,
terpenoids, saponins, tannins, steroids, carotenoids, and resins etc. and most commonly the
terpenoid substance called Cucurbitacins (29). They are highly oxygenated, tetracyclic
triterpenes containing a cucurbitane skeleton characterized as 19-(10→9β)-abeo-10α-lanost-
5ene(30).The plants of this family are mainly grown around the tropics and to some extent in
temperate areas. Some of the important plants that have been extensively studied in this
family are Mukia maderaspatana, Solena amplexicaulis, Citrullus colocynthis, Citrullus
lanatus, Coccinia indica, Cucumis sativus, Cucurbita pepo, Lagenaria siceraria, Luffa
acutangula, Trichosanthes cucumerina, Corallocarpus epigaeus, Luffa cylindrica,
Momordica charantia, Trichosanthes dioica and Kedrostis foetidissima (28).

The various cucurbitacins differ with respect to oxygen functionalities at various positions
(30). Many pharmacological and clinical investigations have verified that cucurbitacin B

3
(CuB) and cucurbitacin E (CuE) possess various pharmacological activities, such as,
antitumor, anti-hepatitis and immunopotentiating effects (29). The cucurbitacin preparation
used clinically contains mostly CuB and CuE, which are obtained from the calyx of
Cucumismelo L., a Chinese medicinal plant that is effective against chronic hepatitis and
primary liver carcinoma (28). Seeds or fruit parts of some cucurbits are reported to possess
purgative, emetic and anthelmintic properties due to the secondary metabolite cucurbitacin
contents (31).
Previous literature has found that Cucurbitaceae family have numerous medicinal properties
such as anti-HIV, anxiolytic, anti-pyretic, anti-diarrheal, carminative, antioxidant, anti-
diabetic, antibacterial, laxative, anti-helminthic, anti-tuberculosis, and purgative. It is also
employed as an abortifacient, diuretic, and cardiotonic agent. They also show strong anti-
inflammatory, antitussive, cytotoxic, and expectorant properties (30).

1.5. The Genus Cucumis

The genus name Cucumis is the Latin name for the cucumber which was already cultivated in
Ancient Egypt (32).The genus Cucumis belongs to the family Cucurbitaceae, order
Cucurbitales is represented by 32 species. According to specific morphological features of
tendrils pollen grains and ovules, there are clear relation of this taxon with the order
Passiflorales (33).

The plants are annual herbs, exceptionally typically having a climbing growth habit or
trailing and semi shrubs, although few cucumber and C. melo cultivars have a root system,
rarely woody (C. trigonus) are extensive, bush habit, but not often tuberous (C.
kalahariensis), usually shallow. Stems are sulcate, angled, not aculeate or rarely aculeate (C.
ficifolius), rarely glabrous or variously pubertal, rarely breakaway hairs or with non-break
away hairs (C. sacleuxii) (34).

The genus Cucumis indigenous mainly to Africa, also Asia, Australia and some islands in the
Pacific. It includes two major commercial vegetable crops: C. sativus (cucumbers, from Asia)
and C. melo (Melons, Africa and Australia, Asia), and two minor ones: The West Indian
gherkin (C. anguria) and the kiwano (C. metuliferus). These last two species became
cultivated crops outside their native Africa (32).

Pharmacological experiment conducted on C. melo and some Cucumis spp., point to its
immense prospective in the management of conditions such as inflammation, pain, cancer,
cough, liver diseases, and cardiovascular disorders (34). Cucumis melo whole fruit is useful

4
in chronic eczema. The fruit is tonic, laxative, and galactagogic, diuretic and diaphoretic. The
flowers used as expectorant and induce vomiting. The seeds are used as cough suppressant,
fever reducer, and a digestive aid. A seed powder is mixed with water and used as anti-
helminthic (30). Cucumis sativus fruit help in removing constipation and ingestion the fruits
are used during summer as cooling food, seeds as anthelmintic (30).

1.5.1. Pharmacological activity of genus Cucumis

Antimicrobial activity
In the previous study the crude extract of the whole plant of Cucumis melo.L (Cucurbitaceae)
was screened for activity against bacterial strains like E. coli, K. pneumoniae, S. paratyphi, S.
aureus and a fungal strain C. barbicans using aqueous, heptane, petroleum ether and acetone
as a solvent by using disk diffusion method. Highest zone of inhibition was shown by whole
plant and fruit extract of cucumis melo L. with aqueous and acetone with C. albicans and E.
coli 08 and 12 mm respectively(35).The previous study on the cucumis sativus using disk
diffusion shows higher antibacterial and antifungal activity against the following
microorganisms like S. typhi, E.coli, E. faecalis, B. cereus and C. lunata, C. albicans. Also, it
justifies. Antimicrobial activities are aggravated by increasing the quantity of this compound,
which can be used as an alternative for antibiotics (36).
Antibacterial and antifungal activity was studied by Agar well diffusion method and was
concluded that the fruit ethanolic extract of Cucumis anguria shows stronger antimicrobial
activity than its methanolic, chloroform, ethyl acetate extracts (37).

Antidiabetic activity
On the basis of previous evidences obtained in laboratory animal study, the researchers
concluded that the Cucumis sativus extract possesses hypoglycemic properties which
suggests the presence of biologically active components such as terpenoids, alkaloids,
flavonoids, and phenolics have shown antidiabetic potential through the insulin mimetic
activity of the plant extract which may be worth further investigation and elucidation (38).
Data show that the extract of Cucumis sativus normalize the blood glucose level of rats.
Hence, it might help in preventing diabetic complications and may serve as a good alternative
of antidiabetic drugs (38).
C. melo leaf methanolic extract was investigated for its antihyperglycemic effects against
streptozotocin induced hyperglycemia in rats and concluded that methanolic extract of C.

5
melo leaf have greater anti-hyperglycemic activity than aqueous extract in streptozotocin
induced hyperglycemia model and when compared with glibenclamide treated group (39).

Antioxidant Activity
Qualitative DPPH spray method was used for checking free radical scavenging activity of
Cucumis anguria extract and free radical in it gives a strong absorption maximum at 517 nm
and purple colour were changed to yellow which can be concluded as extract has anti-
oxidant properties (40). The methanolic extract of Cucumis trigonus roxb seeds gave
concentration-dependent DPPH radical scavenging activity. The highest free radical
scavenging activity was found to be 83.66% at concentration of 200 μg/ml which was
comparable to the free radical scavenging activity of standard (0.05 mM) ascorbic acid (41).
In DPPH assay the percentage of antioxidant activity of Cucumis melo Seed extracts values
recorded at 30 minutes were analyzed. Cucumis melo seed extracts were analysed the highest
radical scavenging activity (73.2 %) and followed by others (42). Another previous study
showed that Response of Cucumis dipsaceus fractional extracts towards various antioxidant
assays was appreciable especially in 2’-azinobis (3-ethylebenzothiozoline-6-sulphonic acid
radical, metal chelating, nitric oxide and DPPH assays. Methanol extract of Cucumis
dipsaceus (10.37 µg/mL) assay revealed higher free radical inhibition (43).

1.5.2.Isolated compound
Preliminary phytochemical screening of the ethanol and crude chloroform extract of leaves
and steam of Cucumis sativus possessed phytoconstituent such as alkaloid, glycoside, steroid,
saponin and tannins where as Cucumis sativus fruits revealed the presence of glycosides,
steroids, flavonoids, carbohydrates and tannins (29). Flavone glycosides such as isovitexin,
saponarin and various acylated flavone C-glycosides are present in the leaves. Antiulcer 9-
beta-methyl-19-norlanosta-5-ene type glycosides have been isolated from Cucumis sativus
Seeds (31). Cucumis melo fruit has a high Superoxide Dismutase which is responsible for the
in vitro and in vivo antioxidant and anti-inflammatory properties of the extract. Furthermore,
number of phenolic glycosides have been isolated from the seed (30). Cucumis anguria
showed to possess antioxidant activity and various constituents Such as flavonoids, alkaloids,
triterpenoids, carbohydrates, tannins, sterols, anthraquinone, glycosides and saponins are
present in it (40). Phytochemical screening of Cucumis metuliferus revealed the presence of
alkaloids, flavonoids, triterpenoid, saponins, steroids, volatile oils, total glycosides, cardiac
glycosides and saponin glycosides (44).

6
The following figure shows some isolated structure from some species of genus Cucumis
(45,46).

Figure 1: Phenylethyl isolated from the Cucumis melo which is taken from Brazilian Journal of
Pharmacognosy on page 465.
where 1: Cucumins, 4 R=H, luteolin ,5R=OH, quercetin; 2: R1=H,R2=R3=OH, 5,7-
dihydroxy-2- [2-(4-hydroxyphenyl)ethyl]chromone; 3: R1=R2=R3=OH,5,7-
dihydroxy-2-[2-(3,4- dihydroxyphenyl)ethyl]chromone 6 R1=H,R2=OH,R3=O-O-
Glu,7- glucosyloxy-5-hydroxy-2-[2-(4-hydroxyphenyl)ethyl]chromone.

Figure 2: Sphingolipids Isolated from the Stems of Cucumis sativas L wohich is taken from journal
of Molecules on page 9290.

7
1: (2S,3S,4R,10E)-2-[(2'R)-2-hydroxytetracosanoylamino]-1,3,4-octadecanetriol-10-ene;
2: 1-O-β-D-glucopyranosyl-(2S,3S,4R,10E)-2-[(2'R)-2-hydroxytetracosanoylamino]-1,3,4-octadecanetriol-10-
ene; 3: soya-cerebroside.

1.6. Cucumis ficifolius

Figure 3: Morphological view of Cucumis ficifolius A. Rich Taken from Gelan T. Antimicrobial
Activity of Solvent-Extracts of Cucumis ficifolius and Zehneria scabra on some Test
Microorganisms. MSc Thesis Addis Ababa University (Unpublished). 2011.on page 56.

1.6.1. Botanical Description

C. ficifolius is a perennial herb climbing to 2m long from a perennial wood root stock up
to1mm long. The stems are hairy with short fine hairs and also with small aculeate hairs of
varying degrees or stoutness; Leaves are blade ovate to broadly rotundate in outline; Seeds
are Elliptic. In Ethiopia, the plant is locally known as Yemidier embuay (Amharic language)).
The plant distributed in different part of Ethiopia mainly Tigray, Go jam, Shewa, Welega,
Gamo Gofa and Harare. This plant mainly growing at the altitude ranging from 1300-2400 m
(47).

1.6.2. Ethnobotanical uses

In Ethiopia, the fruit part reported to treat rabies. And also, fresh fruit used as addressing for
inflamed fingers in Nigeria and Ethiopia (47). In Some places it is an ingredient of medicine
for syphilis and as an emetic and in small doses with honey to relieve stomach ache for
children, in Ethiopia it also used for the treatment of various ailments such as “Kuruba”,
“Chiffea”, “Mageriat geter” (meningitis), “nessir” (epistaxis),“Wefbeshita” (48). And also,
the roots are used for complete treatment of malaria. The seeds are oil giving. The Root
extract of C. ficifolius is recorded to be used in local Honey-wine or “Tej” to make beverage

8
more intoxicating (47). Previous study in Dek island by confirmed that different parts of
cucumis ficifolius used for different ailments such as Stomach-ache, nail injury, wart,
dysentery, ‘mich’, meningitis and evil eye (48).

1.6.3. Pharmacological activity

In the previous investigation, the antimicrobial activity of the chloroform, ethyl acetate,
acetone, ethanol and methanol extracts of the leaves of C. ficifolius were evaluated using disk
diffusion method. Tetracycline and erythromycin were used as positive control whereas
Tween 20 served as a negative control and extracts showed varying degree of inhibitory
activity against S. aureus, E. coli, P. aeruginosa and S. boydii at concentration of 400mg/ml
(47).

1.6.4. Significance of the study

The emergence of multidrug resistant strain of pathogens such as S.aureus, E.coli, E.fecalis,
P.aurginosa, K. Pneumonia andS.pyogens is a serious threat and makes chemotherapy more
difficult. Development of new antimicrobials is among the proposed solutions to curb this
problem. In this regard, plants could provide a good alternative in search for new chemical
agents with a wide-ranging antimicrobial activity and less side effect (17). Screening of
medicinal plants for their bioactivity and phytoconstituents is extremely important to identify
promising candidates that are sources of potential therapeutic agents, which can be used as a
guide for the selection this medicinal plant as a potential species in order to establish further
structural elucidation and structure activity relationship, which lead the way to discover a
novel antimicrobial agent for therapeutic use. To the best of our knowledge, there was no
previous studies have been conducted so far on the antibacterial activities of the extracts of
Cucumis ficifolius A. Rich.

9
2 Objectives

2.1. General objectives

 The aim of this study was to screen the preliminary phytochemical constituents and to
evaluate the in vitro antibacterial activity of activity of the root extracts of Cucumis
ficifolius A. Rich against selected bacterial strains.

2.2. Specific objectives

 To undertake preliminary phytochemical screening of the crude hydro alcoholic
root extract of C. ficifolius.
 To evaluate the antibacterial activity of the crude root extract of C. ficifolius using
agar well diffusion method.

10
3 Material and Methods

3.1. Chemicals
The following drug, chemicals, reagents and solvents were used: Ciprofloxacin (Addis
pharmaceutical factory, Ethiopia), muller-Hinton agar (Oxide Pvt.Ltd., United Kingdom),
NaOH (Supertek chemical reagent Ltd., India), H2SO4 (Himedia laboratories reagent
Ltd,India), HCl (Nice laboratory reagent Ltd, India), methanol, ammonia, ferric chloride,
DMSO, mayer reagent and benzene (Loba Chemie Pvt-Ltd., India), ethanol and chloroform
(Blulux Laboratory (P), India), and wagner Reagent (Research Lab Fine. Chem Ltd., India).

3.2. Equipments and supplies

The following equipment and materials were used: electrical balance, desicator, oven ,
Erlenmeyer flask, filter paper, stirrer, water bath, distelid water, measuring cylinder, pippete,
conical flask, beaker, mortal and pistil, diteregentsoap, test tube, glove facemask, sterilizer,
refrigerator.

3.3. Plant materials

The root of C. Ficifolius was collected from Estie town which has Latitude:11° 39' 59.99" N
, altitude range of 1774-2414meters above see level known by wenadega(sub tropical)
climatic condition and located in South Gondar, Amhara region, in January 2018 Ethiopia,
165 km far from Gondar. Then the Botanical identification and authentication of the plant
materials were performed by Abiyu Eniyewu (botanist) and voucher specimens (No: 00SA-
FM) has been deposited in Herbarium Biology Department, Faculty of Natural and
Computational Science, University of Gondar.

3.4. Bacterial strains

The test was carried out against three gram positive bacterial strains: Staphylococcus aureus
(ATCC 25923), Enterococcus faecalis (ATCC 1912/R) and Streptococcus pyogenes (ATCC
19615) and three gram negative bacterial strains: Escherichia coli (ATCC 25922),
Pseudomonas aeruginosa (ATCC 2706) and Klebsiella pneumonia (ATCC 700603).
Pseudomonas aeruginosa (ATCC 2706), Klebsiella pneumonia (ATCC 700603)and
Enterococcus faecalis (ATCC 1912/R) were obtained from Amhara Regional Laboratory,
Bahr Dar and trasported by bus in side of culture midia stored by ice and were stored in -20
°C together with bacterial strains obtaind from UOG till the actual experment was carried
out.

11
3.5. Preparation of plant extract
The roots were washed thoroughly under tap water and shad dried at room temperature for
three weeks. Thereafter, the dried roots were grinded into a coarse powder and 600 g of
powder was macerated in 80% methanol for the period of 72 h with occasional shaking and
stirring. This was repeated for two times and the extracts thus obtained were filtered two
times through filter paper (Whatman Fitter Paper No. 1). Then, the filtrate was allowed to
evaporate using oven at temperature 40 °C. Finally, a highly concentrated methanol extracts
were obtained and stored in refregeter at less than 80c and while experiment carried out store
in desicator.

3.6. Determination of antibacterial activity

Bacterial broth culture (Oxide Pvt.Ltd., United Kingdom) was prepared to a density of 108
cells ml-1 of 0.5 Mc Farland standards which are used as a reference to adjust the turbidity of
bacterial suspensions so that the number of bacteria will be within a given range to
standardize microbial testing and on each plate; equidistant wells were prepared with a 6 mm
diameter sterilized cork borer then randomly labelled. Then, a liquot was spread evenly onto
Muller-Hinton agar using sterilized cotton swab. Next, 50 μL of each extract at
concentrations of 1, 2 and 4 mg/ml L, by using DMSO as solvent, were applied aseptically
into a respective agar wells. Standardized ciprofloxacin 10 µg/mL, dissolved by DMSO, was
used as positive control and DMSO (50 μL) was included as negative control. The agar plates
were allowed to stand on bench for 30 minutes at room temperature for pre-diffusion and
then incubated at 37oC for 24 hours. Antibacterial activity was determined by measuring the
Zone of inhibition (ZI) around each well (excluding the diameter of the well by subtracting 6
mm from ZI results). For each concentration, three replicate trials were conducted against six
organisms (49).

3.7. Preliminary Phytochemical screening

The crude extract of cucumis ficifolius was screened for the presence or absence of secondary
metabolites such as alkaloids, steroidal compounds, terpenoids, phenolic compounds,
flavonoids, tannins and saponins by using the methods as described by standard procedures.

Test for terpenoids (Salkowski test)

One hundred milligram of the extract was mixed with 2 ml chloroform and 3 ml of sulfuric
acid was added to form a layer. A reddish-brown coloration of the interface was an indication
of terpenoids (50).

12
Test for tannins
A half gram of the dried powdered plant sample was boiled in 20 ml of water in a test tube
and filtered. Few drops of 0.1% ferric chloride were then added to the filtrate and the
formation of brownish green or a blue-black coloration was regarded as indicator of the
presence of tannins (50).

Test for alkaloids

Mayer’s test: To a few ml of filtrate, 2-3drops of Mayer’s reagent (Potassium mercuric
iodide solution) was added along the slides of the test tube. Formation of white or creamy
precipitate indicates presence of alkaloids (51)

Wagner’s test: To a few ml of filtrate, 2-3drops of Wagner’s reagent (Potassium iodide

solution) was added along the sides of the test tube. Formation of reddish brown precipitate
indicates presence of alkaloids (51).

Test for flavonoids

NaOH test: 50mg of extract was dissolved in 2ml of alcohol and to the extract, increasing
amount of NaOH was added. It shows yellow coloration which decolorizes after addition of
an acid (51).

Test for saponins

Foam test: About 50mg of extract was dissolved in 2ml of alcohol, diluted with20ml of
distilled water and shaken for 15min in a graduated cylinder. A layer of stable foam indicates
presence of saponin glycosides (51).

Test for phenols

FeCl3 test: About 50mg of extract was dissolved in 2ml of distilled water, add 2drops of
neutral 5% FeCl3 solution and observed for coloration. Formation of blue, green and violet
colour indicates the presence of phenolic compounds (52).

Test for steroids

Salkowski test: To about 50mg of extract, 2ml of chloroform and 2ml of concentrated
H2SO4 were added and shaken well. Then observed for coloration of CHCl3 and acid layer.
appearance of chloroform layer in red color and acid layer as greenish yellow fluorescence
indicates the presence of steroids (51).

13
Test for anthraquinone
Boil 200 mg of extract with 6 ml of 1% HCl and filtered. Shake the filtrate with 5 ml of
benzene, filtered and add 2 ml of 10% ammonia solution to the filtrate. Then shake the
mixture and the presence of a pink, violet or red colour in the ammoniacal phase indicated the
presence of free hydroxyl anthraquinones (51).

3.8. Ethical consideration

This experiment was conducted as per the internationally accepted guideline and
protocol.(49).
Furthermore, letter of approval was obtained from School of Pharmacy, College of Medicine
and Health Sciences, University of Gondar.

14
 4. Results

4.1 Percentage yield of crude extract

The 80% methanolic crude extracts of the dried root of cucums. Ficifolius (600 gm) gave a
shiny reddish powder of 150 gm. Thus, the percentage yield becomes 25% on dry weight
basis.

4.2. Preliminary phytochemical screening

As shown in Table1, preliminary phytochemical screening of the hydroalcoholic root extract
of Cucumis ficifolius revealed the presence of bioactive compounds such as alkaloids,
steroids, flavonoids, phenols terpinoids and antraqunolones while Alkaloid (mayer’stest),
tannin and saponin are relatively absent.
Table 1: Preliminary phytochemical screening results of the root extract of Cucumis ficifolius

Photochemical group Specific tests Results

Alkaloids Mayer’s Test +
Wagner’s Test +++
Tannins FeCl3 +
Flavonoids Nao H test +++
Phenol Only 1% FeCl3 +++
Anthraquinone BornTrager’s test +++
Steroids Salkowski test) +++
Terpinoid Salkowski test) +++
Saponins Froth Test +
(+): weak; (++): moderate; (+++): strong

15
4.3. Anti-bacterial activity
The antibacterial assay was done for six bacterial strains, three gram positive (S. aureus, S.
pyogens and E. fecalis) and three gram negative (E. coli, P. aurginosa and K. Pneumonia).
Triplicate was done for all six bacterial strains. The standard drug ciprofloxcilln at a
concentration of 10 𝜇g/ml show a potent antibacterial effect against all bacterial strain except
S. pyogen bacterial strain which has zero ZI. However, the methanolic crude extract of C.
ficifolius at three different concentrations (1, 2 and 4 mg/ml) had shown almost zero ZI for all
tested bacteria strains (Table 2). Hence, the crude extract lacks the antibacterial effect at
these concentrations.

Table 2: Antibacterial activity of crude root extracts of Cucumis ficifolius A. Rich

Tested bacteria Etract Hydroalcoholic Ciprofloxacin DMSO (50 μl)

dose(mg/ml) root extract ZI (10 µg/ml) ZI (mm)
(mm)
S. aureus 1 mg /ml 0
ATCC 25923 2 mg /ml 0 33.333 0
4 mg/ml 0
S.pyogenes 1 mg /ml 0 0 0
ATCC 19615 2 mg /ml 0
4 mg/ml 0
E.Faecalis 1 mg/ml 0
ATCC 1912R 2 mg/ml 0 35.666 11.333
4 mg/ml 0
P.aeruginosa 1 mg/ml 0
ATCC 2706 2 mg/ ml 0 39 0
4 mg/ ml 0
K. pneumoniae 1 mg/ml 0
ATCC 700603 2 mg/ ml 0 23.666 0
4 mg/ml 0
E. coli ATCC 1 mg /ml 0
25922 2 mg/ml 0 35.666 10
4 mg/ml 0

16
 5. Discussion
The present study was undertaken to evaluate the antibacterial activity of the root extract of
Cucumis ficifolius A. Rich. Traditionally different part of this plant has been used for the
treatment of various ailments. For instance, the dried root been used for the treatment of skin
disease, swellings in the body, and wound dressings (48). The report of previous study on
anti-microbial activity of the leaf of C. ficifolius A. Rich has confirmed the presence of both
antibacterial and antifungal activities recommend that further investigation for the presence
of secondary metabolites while it has good antibacterial and anti-fungal activity (47). Despite
of plant material has important secondary plant metabolites,lack of effective antibacterial
activity of might be due to degradation of the chemical constituents due to heating in oven for
one month, procedural and methodological limitation , actual lack of antibacterial activity or
error in concetration of the of extract.
Here in this study the negative control (DMSO vehicle) has higher zone of inhibition than
crude extract of cucumis ficifolius A.rich on some tested common pathogenic bacteria. This
result does not need further confirmation by using another solvent (i.e. sterile water) to
confirm whether negative control or extract has anti-bacterial activity as the initial extract
does not show any activity. This is done because the negative control DMSO has some anti-
bacterial activity.The recommended and standard method of antibacterial assay is serial
dilution techniques but in this study there was no satisfactory result in the Agar well
diffusion test it is not necessary to conduct serial dilution test.
In this study the % yield of the extract is high might be due to two times filtration during
maceration. With regard of phytochemical constituent’s root extract revealed that the
presence of alkaloids, flavonoids, steroid, phenolic, anthraquinone and terpenoids. These
secondary plant metabolites have confirmed and versatile medicinal properties which may be
a reason for the use of the plant material for different health problems traditionally. The
finding of this work is encouraging and indicates that the herb should be studied more
extensively for its therapeutic benefits.

17
 6. Conclusion
In conclusion, despite of the previous finding of the leaf extract and ethnobotanical claim of
the root of C. ficifolius, the current search on root part indicated that the absence of
antibacterial activity of different concentration of the crude extract against the tested bacterial
strain. Phytochemical screening of the crude extract reveals the presence of alkaloids,
flavonoids, phenols, steroids, terpinoids and antraqunionens.

18
 7. Recommendation
Based on the present study the following recommendations are proposed.
 Additional study is warranted by using different solvent other than methanol as
different extraction methods for further confirmation/ disproving of the
ethnobotanical claim;
 Further study targeting different bacterial strains other than strains tested in this
experiment is suggested;
 The use of lyophilizer or water vapour instead of oven which may prevent
degradation of thermolabile constituents.
 The use of fresh root instated of dried root may preserve volatile oils which might
have a therapeutic value.
 Future investigation also recommended at high concentration of the plant extract.

19
 8. References
1. Barreto ML, Teixeira MG, Carmo EH. Infectious diseases epidemiology. J Epidemiol
Community Health. 2006;60(3):192–5.
2. Sverrisdóttir BB. Immune modulation in Staphylococcus aureus -induced arthritis by a
combination of antibiotics and inhibition of Interleukin-15. 2012.
3. Pavic S, Brett M, Petric N, Lastre D, Smoljanovic M, Atkinson M, et al. An outbreak of
food poisoning in a kindergarten caused by milk powder containing toxigenic Bacillus
subtilis and Bacillus licheniformis. Arch Lebensmittelhyg. 2005;56(1):20–2.
4. Karch H, Tarr PI, Bielaszewska M. Enterohaemorrhagic Escherichia coli in human
medicine. Int J Med Microbiol. 2005;295(6–7):405–18.
5. Wang S, Zhang Y, An W, Wei Y, Liu N, Chen Y, et al. Magnetic relaxation switch
immunosensor for the rapid detection of the foodborne pathogen Salmonella enterica in
milk samples. Food Control. 2015;55:43–8.
6. Cavallaro E, Date K, Medus C, Meyer S, Miller B, Kim C, et al. Salmonella
Typhimurium Infections Associated with Peanut Products. N Engl J Med [Internet].
2011;365(7):601–10.
7. Fine PEM. The Interval between Successive Cases of an Infectious Disease. Am J
Epidemiol. 2003;158(11):1039–47.
8. Zhang L, Ravipati AS, Koyyalamudi SR, Jeong SC, Reddy N, Bartlett J, et al. Anti-
fungal and anti-bacterial activities of ethanol extracts of selected traditional Chinese
medicinal herbs. Asian Pac J Trop Med. 2013;6(9):673–81.
9. O’Neill J. Tackling drug-resistant infections globally: final report and recommendations.
Rev Antimicrob Resist. 2016;:84.
10. WHO. Global report for research on infectious diseases of poverty. Who [Internet].
2012;1–184.
11. Stanković N, Mihajilov-Krstev T, Zlatković B, Stankov-Jovanović V, Mitić V, Jović J,
et al. Antibacterial and Antioxidant Activity of Traditional Medicinal Plants from the
Balkan Peninsula. NJAS - Wageningen J Life Sci. 2016;78:21–8.
12. Cosgrove SE, Carmeli Y. The Impact of Antimicrobial Resistance on Health and
Economic Outcomes. Clin Infect Dis [Internet]. 2003;36(11):1433–7.
13. Bhat MA, Malik RA, Prakash P, Lone AM. Preparation and evaluation of antibacterial
potential of Pithecellobium dulce root extract against Gram positive and Gram negative
bacteria. Microb Pathog [Internet]. 2018;116:49–53.

20
14. Chandra M. Antimicrobial Activity of Medicinal Plants against Human Pathogenic
Bacteria. Int J Biotechnol Bioeng Res. 2013;4(7):2231–1238.
15. Tittikpina NK, Nana F, Fontanay S, Philippot S, Batawila K, Akpagana K, et al.
Antibacterial activity and cytotoxicity of Pterocarpus erinaceus Poir extracts, fractions
and isolated compounds. J Ethnopharmacol. 2018;212:200–7.
16. Analysis E. Health Monitor. 2012;2016.
17. Pharcomagnosy and Phytotherapy. 2014;6(2).
18. Kurmukov AG. Phytochemistry of medicinal plants. Med Plants Cent Asia Uzb Kyrg.
2013;1(6):13–4.
19. Khan R, Islam B, Akram M, Shakil S, Ahmad A, Ali SM, et al. Antimicrobial activity
of five herbal extracts against Multi Drug Resistant (MDR) strains of bacteria and fungus
of clinical origin. Molecules. 2009;14(2):586–97.
20. Mambe FT, Voukeng IK, Beng VP, Kuete V. Antibacterial activities of methanol
extracts from Alchornea cordifolia and four other Cameroonian plants against MDR
phenotypes. 2016;11(2):121–7.
21. Klančnik A, Piskernik S, Jeršek B, Možina SS. Evaluation of diffusion and dilution
methods to determine the antibacterial activity of plant extracts. J Microbiol Methods.
2010;81(2):121–6.
22. Sibanda T, Okoh a. I. The challenges of overcoming antibiotic resistance: Plant
extracts as potential sources of antimicrobial and resistance modifying agents. African J
Biotechnol. 2007;6(25):2886–96.
23. Lewis K, Ausubel FM. Prospects for plant-derived antibacterials. Nat Biotechnol.
2006;24(12):1504–7.
24. Yu T, Yamaguchi H, Noshita T, Kidachi Y, Umetsu H, Ryoyama K. Selective
cytotoxicity of glycyrrhetinic acid against tumorigenic r/m HM-SFME-1 cells: Potential
involvement of H-Ras downregulation. Toxicol Lett. 2010;192(3):425–30.
25. Cushnie TPT, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimicrob Agents.
2005;26(5):343–56.
26. Scalbert A, Williamson G. Chocolate : Modern Science Investigates an Ancient
Medicine Dietary Intake and Bioavailability of Polyphenols 1. J Nutr [Internet].
2000;130((8S Suppl)):2073–85.
27. Claudine Manach, Augustin Scalbert, Christine Morand, Christian Rémésy and LJ.
Polyphenols - Food Sources and Bioavailability.pdf. Am J Clin Nutr [Internet].
2004;79(5):727–47.

21
28. Jamuna S KK and PS. Phytochemical and pharmacological properties of certain
medicinally important species of Cucurbitaceae family – a review. J Res Biol.
2015;5(6):1835–49.
29. Rajasree RS, Sibi PI, Francis F, William H. Family – A ReviewPhytochemicals of
Cucurbitaceae. 2016;8(1):113–23.
30. Saboo SS, Thorat PK, Tapadiya GG, Khadabadi SS. Ancient and Recent Medicinal
Uses of Cucurbitaceae Family. Int J Ther Appl. 2013;9(2013):11–9.
31. Rajasree RS, Sibi PI, Francis F, William H. Phytochemicals of cucurbitaceae family –
A review. Int J Pharmacogn Phytochem Res. 2016;8(1):113–23.
32. Kwaghe A, Ministry F, Devel R. Uses of Cucumis metuliferus : A Review.
2015;(November 2016).
33. Krístkova E, Lebada A, Vinter V, Blahousek O. Genetic resources of the genus
Cucumis and their morphological description. Hortic Sci. 2003;30(1):14–42.
34. Vishwakarma VK, Gupta JK, Upadhyay PK. Pharmacological importance of Cucumis
melo L.: An overview. Asian J Pharm Clin Res [Internet]. 2017;10(3):8–12.
35. Access o. Antimicrobial and antifungal activity of cucumis melo l . ( cucurbitaceae )
and pergularia daemia frosk . ( asclepiadaceae ) an ethnomedicinal plants. 2014;3661–5.
36. Nadu t, nadu t. Research article antimicrobial activity of. 2016;36(16):97–100.
37. On arjouaroppoc, properties p, cucumis of. International journal of universal a review
on pharmaceutical properties of cucumis. 2014;3(june):508–13.
38. Ml B. Hypoglycemic Activity of. 1994;17(9):1026–30.
39. Asif HM, Akhtar N, Sultana S, Rehman SU, Akram M, Rehman U. ISSN 2311-4673
Journal of Pharmacy and Pharmaceutical Sciences Medicinal Properties of Cucumis melo
Linn . 2014;2(1).
40. Gill ns, mahajan a, arora r. Therapeutic potential. 2014;4(07).
41. Snp N, Cucumis OF, Extract M. International Journal of Universal. 2014;3:392–6.
42. Arputhadas G. Family : Cucurbitaceae.
43. Nivedhini V, Chandran R, Parimelazhagan T. Chemical composition and antioxidant
activity of Cucumis dipsaceus Ehrenb. Ex Spach fruit. Int Food Res J. 2014;21(4):1465–
72.
44. Aliero AA, Gumi AM. Studies on the germination, chemical composition and
antimicrobial properties of Cucumis metuliferus. Ann Biol Res [Internet].
2012;3(8):4059–64.
45. Tang J, Meng X, Liu H, Zhao J, Zhou L, Qiu M, et al. Antimicrobial activity of

22
 sphingolipids isolated from the stems of cucumber (Cucumis sativus L.). Molecules.
2010;15(12):9288–97.
46. Ibrahim SRM, Mohamed GA. Cucumin S, a new phenylethyl chromone from Cucumis
melo var. Reticulatus seeds. Brazilian J Pharmacogn 2015;25(5):462–4.
47 Gelan T. Antimicrobial Activity of Solvent-Extracts of Cucumis ficifolius and Zehneria
scabra on someTest Microorganisms.MScThesis Addis Ababa University (Unpublished).
2011.

48. Teklehaymanot T. Ethnobotanical study of knowledge and medicinal plants use by the
people in Dek Island in Ethiopia. 2009;124:69–78.
49. Chauhan JB. A c a d e m i c S c i e n c e s. 2012;4(3):4–6.
50. Edeoga HO, Okwu DE, Mbaebie BO. Phytochemical constituents of some Nigerian
medicinal plants. African J Biotechnol [Internet]. 2005;4(7):685–8.
51. Qadir U, Paul VI, Ganesh P. Preliminary phytochemical screening and in vitro
antibacterial activity of Anamirta cocculus ( Linn .) seeds. 2015;27(2):97–104.
52. Van Middlesworth F, Cannell RJP. Dereplication and Partial Identification of Natural P.
1998. 279-327
53. Jones WP, Chin Y, Kinghorn AD. The role of pharmacognosy in modern medicine and
pharmacy. Curr Drug Targets. 2006;7(3):247–64.

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 9. Annex

Filtration Process Extract in in desicator Diution of extrat during

anti bacterial test

Measuring of zone of inhibition

By Samuel Agegnew phytochemical screening

24